The overall direction of the Molecular Mechanisms of Tumor Promotion Section is to elucidate the mechanisms of action of the phorbol esters and their endogenous analog, the lipophilic second messenger sn-1,2-diacylglycerol. Protein kinase C (PKC) is the major receptor for these compounds, and our emphasis is correspondingly directed at this family of isozymes. In a collaborative effort, we seek to combine mutational analysis with computer modeling and chemical synthesis to probe ligand - PKC interactions. An important concept to emerge is that different ligands interact through a combination of common and unique contacts. A related issue is the significance of twin phorbol ester binding domains in typical PKCs. By mutating the individual binding domains, we find that the two binding domains of PKC delta are not equivalent; rather, the second domain plays the predominant role in translocation of PKC to the membrane in the presence of phorbol ester. For PKC alpha, in contrast, occupancy of both domains appears essential for translocation, and the rate of translocation depends on the binding affinities of the two domains. Our long term objective is to exploit such isotype differences to dissect subpathways of PKC mediated signal transduction. The bryostatins, although activators of PKC in vitro, function as partial antagonists in intact cells. Consistent with their possessing additional sites of action other than PKC, we find that growth inhibition of B16 melanoma cells by bryostatin 1 does not depend on PKC; rather, derivatives modified to bind PKC only weakly still maintain their growth inhibitory activity. The phorbol-related diterpene resiniferatoxin acts as an ultrapotent analog of capsaicin and has permitted characterization of specific capsaicin receptors. We are now able to define distinct receptor subclasses with distinct functions. In particular, the resiniferatoxin selective subclass is involved in desensitization whereas the capsaicin selective subclass is involved in acute responses. These two subclasses, furthermore, use different signaling pathways. These findings have important implications for the therapeutic development of vanilloids in the treatment of pain.